Bile acid sulfate sulfatase gene, plasmid containing said gene a

Details Bile acid sulfate sulfatase gene, plasmid containing said gene a Bile acid sulfate sulfatase gene, plasmid containing said gene a

1993-10-26

1996-12-17

Wax, Robert A.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

4353201, 536 232, C12N 916, C12N 1555, C12N 1563

Patent

active

055852556

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD

The present invention relates to bile acid sulfate sulfatase and a gene therefor, a plasmid containing the gene coding for said protein, a transformant capable of producing bile acid sulfate sulfatase, and a method of producing bile acid sulfate sulfatase.


BACKGROUND ART

The present inventor previously searched for an enzyme capable of efficiently hydrolyze 3.alpha.-sulfuric acid esters of sulfated bile acids for the purpose of enabling enzymatic assay of sulfated bile acids in blood or urine. As a result, it was found that the bacterial species Pseudomonas testosteroni, which belongs to the genus Pseuomonas, produces the desired enzyme, bile acid sulfate sulfatase. The bile acid sulfate sulfatase produced by said bacterial species is characterized in that it acts on 3.alpha.-sulfated bile acids, leading to the formation of 3.beta.-hydroxy bile acids. Therefore, it was made possible to assay 3.alpha.-sulfated bile acids by oxidizing said 3.beta.-hydroxy bile acids to 3-oxobile acids under the action of .beta.-hydroxysteroid dehydrogenase in the presence of .beta.-NAD, which is a coenzyme for said dehydrogenase, with simultaneous reduction of .beta.-NAD to NADH, and assaying the thus-formed NADH by a per se known method (cf. Japanese Unexamined Patent Publication No. 02-145,183).
However, such a method of producing bile acid sulfate sulfatase as mentioned above has drawbacks. Thus, it is an indispensable condition that cholic acid or the like, which is expensive, should be added, as an inducer substrate, to the medium. Moreover, the yield of bile acid sulfate sulfatase is low and the production procedure is rather complicated.
Accordingly, it is an object of the present invention to provide a method of producing 3.alpha.-bile acid sulfate sulfatase in high yields and at a low cost in an easy and simple manner.


BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a restriction enzyme map of a recombinant plasmid named pABS106.
FIG. 2 shows a restriction enzyme map of a recombinant plasmid named pABS101.
FIG. 3 shows the complete base sequence of the bile acid sulfate sulfatase gene (SEQ ID NO:1).
FIG. 4 shows the amino acid sequence of the peptide obtained by translation of the bile acid sulfate sulfatase gene (SEQ ID NO:2).


DISCLOSURE OF THE INVENTION

The present inventor found that when a transformant (e.g. Escherichia coli) obtained by introduction of a recombinant DNA constructed by inserting, into a vector DNA (e.g. plasmid vector), a 2.36 kb DNA fragment containing the bile acid sulfate sulfatase gene region (1,509 bp) derived from a bile acid sulfate sulfatase-producing bacterial strain belonging to Pseudomonas testosteroni, for example Pseudomonas testosteroni ATCC 11996, is reared and cultivated in an ordinary nutrient medium, said enzyme can be produced efficiently in said transformant without adding any expensive substance such as mentioned above to the medium. The present invention has been completed based on this and other findings.
Thus, the present invention provides a bile acid sulfate sulfatase gene derived from bacteria belonging to Pseudomonas testosteroni and containing a DNA sequence coding for an amino acid sequence of the following formula (A) (SEQ ID NO:2): ##STR1##
The invention also provides a bile acid sulfate sulfatase gene containing a DNA sequence of the following formula (B) (SEQ ID NO:1): ##STR2##
The invention further provides a plasmid containing a DNA sequence coding for the amino acid sequence of the above formula (A) as derived from bacteria belonging to Pseudomonas testosteroni.
Still further, the invention provides bacteria belonging to Escherichia coli and harboring a plasmid containing a DNA sequence coding for the amino acid sequence shown by the above formula (A) as derived from bacteria belonging to Pseudomonas testosteroni.
Furthermore, the invention provides a method of producing bile acid sulfate sulfatase which comprises cultivating in a medium bacteria belonging to the genus Escherichia that have acquired the ability to produce bile acid su

REFERENCES:
patent: 5091305 (1992-02-01), Sugimori et al.
patent: 5100795 (1992-03-01), Sugimori et al.
Hunkapiller et al., Meth. Enzymol. 91:227-236 (1983).
Lathe, J. Mol. Biol. 183:1-12 (1985).
Mashige et al., Clin. Chem. 27:1352-1356 (1981).

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